Systemic fungal infections have become an increasing problem over the last three decades (see, e.g., Bartlett (1991) Clin. Microbiol. Rev. 4:137-149; Bodey et al. (1992) Eur. J. Clin. Microbiol. Infect. Dis. 11:99-109; Sternberg (1994) Science 266:1632-1634; Cox (1993) Curr. Opin. Infect. Dis. 6:422-426; Fox (1993) ASM News 59:515-518; Kujath (1992) Mycoses 35:225-228; Samonis and Bafaloukos (1992) In Vivo 6:83-194, Nouza (1992) Infection 20:113-117). The most serious infections occur in the immunocompromised host and can result in disseminated systemic mycoses (see, e.g., Bartlett (1991) Clin. Microbiol. Rev. 4:137-149; Bodey et al. (1992) Eur. J. Clin. Microbiol. Infect. Dis. 11:99-109; Sternberg (1994) Science 266:1632-1634; Cox (1993) Curr. Opin. Infect. Dis. 6:422-426; Fox (1993) ASM News 59:515-518; Kujath (1992) Mycoses 35:225-228; Samonis and Bafaloukos (1992) In Vivo 6:83-194,Nouza (1992) Infection 20:113-117).
Current treatment for systemic fungal infections is primarily limited to two groups of drugs: the polyene macrolide antibiotics, such as Amphotericin B and nystatin; and the imidazoles, such as ketaconazole and miconazole. Toxicity and resistance to the drugs in use necessitates the discovery and development of new antifungal products.
Zeamatin, a plant protein isolated from corn, has been demonstrated to have antifungal activity in vitro against numerous human pathogens including Candida albicans. (See, Roberts and Selitrennikoff (1990) J. Gen. Microbiol. 136:1771-1778; Roberts el al. U.S. Pat. No. 5,521,153, issued May 28, 1996; Vigers and Selitrennikoff (1991) Mol. Plant Microbe Interac. 4:315-323; Vigers et al. (1992) Plant Sci. 83:155-161). It has been reported that zeamatin, a permatin, acts to inhibit fungi by permeabilizing the plasma membrane, causing the release of the contents of the cell (Roberts and Selitrennikoff (1990) J. Gen. Microbiol. 136:1771-1778).
In addition to its own antifungal activity, zeamatin acts synergistically with a number of antifungal compounds including clotrimazole, Amphotericin B, ketoconazole, grisefulvin, nystatin and nikkomycin X and Z. (See, Roberts and Selitrennikoff (1990) J. Gen. Microbiol. 136:1771-778; Roberts et al. U.S. Pat. No. 5,521,153, issued May 28, 1996; Vigers and Selitrennikoff (1991) Mol. Plant Microbe Interac. 4:315-323). For example, zeamatin enhances the antifungal activity of nikkomycin X and Z against Candida between 100-1000 fold, and can enhance the activity of Amphotericin B against yeast by approximately 3 fold.
Due to its antifungal activity and its synergistic effect on certain antifungal agents, zeamatin may be a useful agent in the treatment of systemic fungal infections. In particular, co-administering zeamatin with other antifungal agents may permit the use of smaller doses of the antifungal agent, thereby reducing the potential for toxic side effects of those agents.
Given the potential for the use of zeamatin, it is desirable to have a high-yield procedure for isolating and purifying the protein on a large scale. The reported method for isolating and purifying zeamatin, illustrated in FIG. 1, uses an ammonium sulfate precipitation step, followed by centrifugation and diafiltration, to obtain a crude protein fraction. This fraction is then further purified by ion-exchange chromatography on CARBOXYMETHYL-SEPHADEX.TM., a cation exchange dextran resin which can be purchased from Sigma Chemical Company or Pharmacia Biotech. The proteins are eluted from the column in a salt gradient, and fractions containing zeamatin activity are pooled. The pooled fractions are subjected to diafiltration using a 10 kDa nominal-molecular-weight cutoff filter, then subjected to reverse phase chromatography using a C-18 resin. Prior art methods for zeamatin purification are described in U.S. Pat. Nos. 5,521,153 and 5,559,034, each of which is specifically incorporated herein by reference in their entirety. These methods are performed on a small scale and are not amenable to scale-up.
The object of the instant invention is to provide a simple, rapid and efficient method for the purification of zeamatin that does not require the time-consuming ammonium sulfate precipitation and centrifugation steps and is amenable to scale-up. Another object of the present invention is to identify column conditions that allow the use of high water content eluents.